This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Formula display:

Abstract

Background

Microbial transformation of steroids has been extensively used for the synthesis of
steroidal drugs, that often yield novel analogues, not easy to obtain by chemical
synthesis. We report here fungal transformation of a synthetic steroidal drug, exemestane,
used for the treatment of breast cancer and function through inhibition of aromatase
enzyme.

Results

Microbial transformation of anti-cancer steroid, exemestane (1), was investigated by using two filamentous fungi. Incubation of 1 with fungi Macrophomina phaseolina, and Fusarium lini afforded three new, 11α-hydroxy-6-methylene-androsta-1, 4-diene-3,17-dione (2), 16β, 17β-dihydroxy-6-methylene-androsta-1, 4-diene-3-one (3), and 17β-hydroxy-6-methylene-androsta-1, 4-diene-3, 16-dione (4), and one known metabolites, 17β-hydroxy-6-methylene-androsta-1, 4-diene-3-one (5). Their structures were deduced spectroscopically. Compared to 1 (steroidal aromatase inactivator), the transformed metabolites were also evaluated
for cytotoxic activity by using a cell viability assay against cancer cell lines (HeLa
and PC3). Metabolite 2 was found to be moderately active against both the cell lines.

Conclusions

Biotransformation of exemestane (1) provides an efficient method for the synthesis of new analogues of 1. The metabolites were obtained as a result of reduction of double bond and hydroxylation.
The transformed product 2 exhibited a moderate activity against cancer cell lines (HeLa and PC3). These transformed
products can be studied for their potential as drug candidates.

Keywords:

Graphical abstract

Background

Microbial transformation of steroids has been extensively employed for the synthesis
of steroidal drugs, both at laboratory and industrial levels [1-7]. In modern drug discovery process, generation of libraries of bioactive compounds
with diverse structures plays an important role [8].

Exemstane (trade name aromasin) is a steroidal irreversible aromatase inhibitor, used
for the treatment of breast cancer. Breast cancers have estrogen receptors (ER-positive)
and their growth depends on aromatase activity. Therefore, inhibition of aromatase
enzyme reduces the estrogen levels and thus slows the growth of breast cancer [9-12].

Interestingly exemestane not only increases the testosterone level and lowers estrogen,
but it also increases the levels of insulin-like growth factor (IGF) [10]. The large reduction in estrogen levels combined with a rise in IGF, makes exemestane
an effective breast cancer medication [13-15]. Based on the importance of exemestane in the treatment of breast cancer, a number
of exemestane derivatives were previously synthesized involving modification of C-6
methylene and reduction of C-17 keto group, and evaluated for their aromatase inhibitory
potential [16-19].

During current study, we synthesized new analogues of 1 by biotransformation techniques.
Screening experiments showed that Macrophomina phaseolina and Fusarium lini were able to efficiently transform 1 into several metabolites. Subsequent large scale fermentations produced three new
metabolites 2-4 along with a known metabolite 5. The structures of metabolites were unambiguously established through detailed spectral
analysis. The microbial transformed metabolites 2 and 4 of exemestane showed a moderate anti-cancer effect against PC3 and/or Hela cancer
cell lines. This successful attempt to synthesize new derivatives of an anti-cancer
steroid may lead to the discovery of new cancer therapeutic agents.

Results and discussion

Four microbial metabolites were generated by the selected fungal strains, i.e. Macrophomina phaseolina and Fusarium lini (Figures 1 and 2). M. phaseolina is previously reported to catalyze the introduction of double bond between C-1 and
C-2, hydroxyl groups at C-6, C-15, C-16 and C-17, and carbonyl group at C-17 of the
steroidal skeleton [1,20]. F. lini is also reported to catalyze the oxidation at C-1, C-2, C-6, and C-11 of steroidal
skeleton [21]. The chemical structures of the metabolites 2-4 are reported here for the first time along with their NMR data (Tables 1 and 2).

Molecular formula C20H24O3 (M+m/z 312.1725, calcd 312.1720) was deduced from the HREI-MS of metabolite 4. A distinct downfield methine proton signal appeared at δ 3.77 (br. s, W1/2 = 9.3 Hz) in the 1H-NMR spectrum of 4. The 13C-NMR spectrum showed a saturated ketone carbon signal at δ 217.7. The rest of the
spectrum was distinctly similar to metabolite 2. The deshielded methine proton was HMBC correlated with this ketonic carbon, while
its corresponding methine carbon at δ 86.8 showed the HMBC correlations with H2-15 (δ 1.95, 2.29), and CH3-18 (δ 0.81). These interactions, along with appearance of a downfield proton (δ 3.77),
indicated that the ketone at C-17 has been reduced into an –OH. Geminal H-17 (δ 3.77)
showed NOESY correlations with H-14 (δ 1.63), indicating it to be axially (α-) oriented. The saturated ketone carbon (δ 217.7) was place at C-16, based on
the above mentioned HMBC correlations (Figure 5). The structure of metabolite 4 was finally identified as 17β-hydroxy-6-methylene-androsta-1, 4-diene-3, 16-dione.

Metabolite 5 has a molecular composition C20H26O2 (HREI-MS, M+m/z 298.1730, calcd 298.1733). Based on 1H- and 13C-NMR spectral data (Tables 1 and 2), compound 5 was identified as 17β-hydroxy-6-methylene-androsta-1, 4-diene-3-one. It has previously
been reported as an in-vitro cytochrome P450-mediated transformed product of exemestane [22].

The cytotoxic effect of the compounds 1-5 against two tumor cell lines, PC-3 (prostate cancer cell) and Hela (cervical cancer
cell), was evaluated (Table 3) using the MTT assay. Compound 2 showed a moderate cytotoxicity against both the cancer cell line with IC50 = 16.83 ± 0.96 and 24.87 ± 0.72 μM, respectively, as compared to the standard drug,
doxorubicin. Compound 4 exhibited a moderate activity against HeLa cell line.

Conclusion

In conclusion, the biotransformation of exemestane (1) with F. lini and M. phaseolina were investigated for the first time which provided an efficient route towards the
synthesis of several new metabolites 2–5. Metabolite 2 was found to be moderately active against both cancer cell lines (HeLa and PC3).
The work presented here can be helpful for the study of in vivo metabolism of exemestane (1), as well as for the discovery of new anticancer drugs

Experimental

Substrate and chemicals

Exemestane (1) was purchased from local market as drug (Pfizer Canada Inc., Brand name Aromasin),
extracted and further purified by flash chromatography. Thin layer chromatography
(TLC) was carried out on silica gel precoated plates (PF254; Merck). Column chromatography (CC) was performed by using silica gel (E. Merck,
Germany). Optical rotations were measured in methanol with a JASCO P-2000 polarimeter.
1H- and 13C-NMR spectra were recorded in (CD3)2CO and CD3OD on Bruker Avance spectrometers. The chemical shifts (δ values) are presented in
ppm and the coupling constants (J) are in Hz. For 1D- and 2D-NMR experiments, standard Bruker pulse sequences were
used. UV Spectra (in nm) were recorded in methanol with a Hitachi U-3200 spectrophotometer.
Infrared (IR) spectra (in cm-1) were recorded with an FT-IR-8900 spectrophotometer. JEOL (Japan) JMS-600H mass spectrometer
was used for recording of EI-MS and high-resolution mass spectra (HREI-MS) in m/z (rel. %).

Cell lines

General Fermentation and Extraction Conditions

4 Liters fungal media was prepared and distributed into 40 conical flasks (100 mL
in each flask). All flasks were then autoclaved at 121°C. The fungal cultures were
then inoculated into each flask containing media and incubated at room temperature
on shaker for three days. Compound 1 was dissolved in 40 mL methanol and distributed equally to all 40 flasks. All experimental
flasks were then kept for fermentation. Two control experiments, i.e. media + compound
1 and media + fungus were also conducted. The transformation was then checked on TLC.
After the detection of transformation on TLC, fungal culture from all 40 flasks was
filtered and extracted with CH2Cl2 (12 L) by using liquid-liquid chromatography. The dichloromethane layer was evaporated
in vaccue. The obtained gum was analyzed by thin-layer chromatography.

Cell Viability Assay

The cytotoxicity of metabolites 1-5 were determined by using MTT-based colorimetric assay in 96-well plate [23]. Both cell lines (PC-3 and HeLa) were cultured in DMEM and MEM media, respectively,
in 25 cm3 tissue culture flasks. The media were supplemented with FBS (5%), pencillin (100
IU/mL) and streptomycin (100 mg/mL). The flasks were then incubated at 37°C in an
incubator containing 5% CO2. The flask (80% confluence) was processed for MTT-based cytotoxicity assay. The percent
viability of the cells was monitored by trypan blue dye. The cells with clear cytoplasm
were considered viable. For the assay, the cells (1 × 105) were loaded onto 96-well tissue culture treated plate. The plate was incubated for
24 hours at 37°C. After incubation, the cells were treated with different concentrations
(1.56-50 μM dissolved in DMSO) of compounds 1-5 and kept in an incubator for 48 hours at 37°C. At the end of the incubation, the
MTT dye (50 μL, 2 mg/mL) was added to each well and the plate was incubated for 4
hours at 37°C in an incubator. Following incubation, the insoluble formazan crystals
were dissolved by adding DMSO (100 μL).

The following formula was used to analyze the cytotoxic effects of the compounds.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

EB, MIC, AR, DF, and CM participated in experimental strategy design, supervision
and manuscript writing. MB and MAI carried out the experiments. AW performed NMR experiments,
while SAS carried out the biological screenings. All authors read and approved the
final manuscript.

Acknowledgements

We would like to acknowledge the Lebanese CNRS (Lebanese Council for Scientific Research)
for a research grant.